Copper resistance in the cold: Genome analysis and characterisation of a PIB‐1 ATPase in Bizionia argentinensis

Abstract Copper homeostasis is a fundamental process in organisms, characterised by unique pathways that have evolved to meet specific needs while preserving core resistance mechanisms. While these systems are well‐documented in model bacteria, information on copper resistance in species adapted to cold environments is scarce. This study investigates the potential genes related to copper homeostasis in the genome of Bizionia argentinensis (JUB59‐T), a psychrotolerant bacterium isolated from Antarctic seawater. We identified several genes encoding proteins analogous to those crucial for copper homeostasis, including three sequences of copper‐transport P1B‐type ATPases. One of these, referred to as BaCopA1, was chosen for cloning and expression in Saccharomyces cerevisiae. BaCopA1 was successfully integrated into yeast membranes and subsequently extracted with detergent. The purified BaCopA1 demonstrated the ability to catalyse ATP hydrolysis at low temperatures. Structural models of various BaCopA1 conformations were generated and compared with mesophilic and thermophilic homologous structures. The significant conservation of critical residues and structural similarity among these proteins suggest a shared reaction mechanism for copper transport. This study is the first to report a psychrotolerant P1B‐ATPase that has been expressed and purified in a functional form.


FIGURE S2.
Multiple alignment section between E. coli CueO (P36649), E.coli PcoA (Q47452) and B. argentinensis Multicopper oxidase (G2EDV6).Conserved residues at the copper binding site are highlighted in blue (Roberts. et al. 2002,Bhamidimarri et al. 2021) and important residues (methionines) at the entrance to the copper site are highlighted in green.

FIGURE S3
. N-terminal portion of the alignment between E. coli CutF (A7ZHT3) and the putative B. argentinensis chaperone (G2ED03).Conservation of the copper-binding site motif (CXXC) is shown in blue, and the periplasmic peptide signal in orange (Öztürk et al. 2021).Complete alignment indicates 35% identity and 52% sequence similarity.

FIGURE S4
. TM helix sequence analysis of P IB -ATPases from B. argentinensis.The alignment of helices TM4, TM5 and TM6 of the four P IB -ATPases (G2EDW3, G2EGV6, G2EC62, G2EHA4) from B. argentinensis is shown.The ion specificity of P IB -ATPases can be predicted from the conserved motif present in each sequence (Argüello 2003).The conserved motif of the IB-1 subtype is highlighted in red bold and the subtype IB-4 is highlighted in black bold.As a result, the proteins can be classified as follow: G2EDW3, G2EGV6, G2EC62 are P IB-1 -ATPases (BaCopA1, BaCopA2 and BaCopA3) and G2EHA4 is a P IB-4 -ATPase (BaCzcP).

FIGURE S5. Schematic representation of transcriptional units found in B. argentinensis
genome containing open reading frames of copper homeostasis related proteins.Potential P IB-1 -ATPase genes are located either within the same transcriptional unit (TU) or in close proximity to other genes associated with copper homeostasis.Specifically, BaCopA1 is part of TU 953, along with the potential periplasmic chaperone (BaCopZP1) and subunit B of the RND transporter (CusB).Upstream TU 953 there are TUs coding for azurin (Azu), a multicopper oxidase (Mco), and subunit A of the RND transporter (cusA).BaCopA2 is located in TU 1522 far away from other genes associated with copper resistance, but close to TU 1519 where a two component regulation system is coded (HK: Histidine kinase, RTF: Regulator transcription factor).The gene encoding BaCopA3 is in TU 618, followed downstream by other TU with genes encoding for the cytochrome oxidase c accessory protein (CcoG), a copper entry transporter (OprC), and an auxiliary chaperone (NosL).Note: The sequences of bacterial proteins involved in copper ions homeostasis were used as seed for Blast-P search of similar proteins on the B. argentinensis genome using NCBI server.This table shows all, the positive and negative results of the blast search.When positive results were obtained, further analysis was done to confirm the presence of essential motifs, signal peptides, membrane regions and others.After that, the proteins of B. argentinensis possibly related to copper management were selected and described in the manuscript.Note: Each pair of sequences were analyzed with Lalign Pairwise Sequence Alignment (PSA) server (Huang and Miller 1991) in order to find the identity and similarity percentage (%).Each CopA from B. argentinensis (their Uniprot code and name are shown) was compared to LpCopA (Uniprot code Q5ZWR1) and AfCopA (Uniprot code O29777).

TABLE S3.
BaCopA1 ATPase activity values corresponding to the conditions depicted in the panels of Figure 5.
Note: The values are obtained from the slope of each curve and normalized by protein mass.

FIGURE S6 .
FIGURE S6.BaCopA1 solubilization assay.Recombinant BaCopA1 was expressed in S. cerevisiae and the membrane fraction was obtained following the protocol described in Experimental Procedures.SDS-PAGE analysis of total membranes (lane 2) and detergent-solubilized membranes (lane 3) is shown in Panel A. The pre-stained molecular weight markers are shown in lane 1, and the corresponding molecular weights are indicated in kDa.Panel B shows the image of the same gel before staining with Coomassie blue and trans illuminated with UV-vis light.A band corresponding to the molecular weight of BaCopA1 (plus GFP) is observed both in total membranes and in detergent-solubilized membranes.Panel C shows the fluorescence spectrum of a sample of 20 μl of total membranes resuspended up to 200 μl in YSB 1X (solid line) and the same volume of membranes solubilized with DDM and resuspended up to 200 μl in YSB 1X (dashed line) upon excitation at 425 nm.These results indicate that BaCopA1 is properly localized in the membrane fraction and that the selected detergent is able to solubilize nearly 80% of BaCopA1 from the membranes.
FIGURE S8.Structural alignments between models and experimental structures.Panel A shows the model for BaCopA1-E1 (magenta) aligned with the experimental structure of AfCopA-E1 (7R0G, gray).Panel B includes the alignment between the model for BaCopA1-E2P (green) and the experimental structure of LpCopA-E2P (4BBJ, gray).Panel C shows the aligned structures of the model BaCopA1-E2Pi (cyan) and those experimentally determined for LpCopA-E2Pi (3RFU, gray).

TABLE S1 .
Reference proteins used as seed for B. argentinensis genome Blast-P search and the results obtained with their corresponding uniprot entry.

TABLE S2 .
Identity and similarity of the aminoacid sequences of BaCopA1, BaCopA2 and BaCopA3 with the sequences of LpCopA and AfCopA.